SUMMARY The spatial and temporal organization of intracellular components by the microtubule cytoskeleton is required for cell division, many aspects of development, and neuronal function. Defects in the microtubule cytoskeleton cause neurological diseases in humans. The long-term goal of this research program is to determine how the multiple components of this transport system?the motors and their tracks, cargos, cargo adaptors, and regulators?work together. Microtubules are dynamic polar structures, with ?plus? ends usually located near the cell periphery and ?minus? ends embedded in internal microtubule organizing centers. Dyneins move towards the minus ends of microtubules, whereas most kinesins move in the opposite direction. In humans, a single dynein (cytoplasmic dynein-1) and ~15 kinesins are responsible for the interphase transport of organelles, proteins, and mRNAs. Viruses hijack these same motors. How does a small subset of motors transport such a large and diverse set of cargos? Understanding this is one of the major frontiers in the transport field. The goal of this proposal is to determine how cargo specificity is achieved for cytoplasmic dynein-1, the major minus- end-directed microtubule-based motor in eukaryotic cells. Traditional biochemical approaches have yielded surprisingly little information about these mechanisms. To solve this problem, we are using both genetic and proteomic discovery approaches to identify motor-cargo interactions and to determine how they are regulated. Genetic approach: Using a forward genetic screen in the filamentous fungus Aspergillus nidulans we co- discovered that peroxisomes ?hitchhike? on early endosomes to achieve motility. Previously, the paradigm was that each cargo directly recruited the transport machinery. We will investigate the mechanism of hitchhiking and determine if it is widely used for organelle motility across eukaryotes. Proteomic approach: Mammalian dynein requires the dynactin complex and a coiled coil containing ?activator? to achieve processive motility. Using proximity-dependent biotinylation we identified the components of the human dynein proteome in human interphase embryonic kidney cells, including novel dynein activators. We will use these proteomic approaches to identify additional novel dynein activators in different cell types and at different stages of the cell cycle. We will use these novel dynein activators as ?stepping stones? to identify their proteomes and determine which cargos they transport.